Abstract:

A high performance liquid chromatographic method with fluorescence detection and pre-column derivatization (HPLC-FLD) has been developed for the determination of seven biothiols including Cys, GSH, and phytochelatins (PCs: PC₂, PC₃, PC₄, PC₅ and PC₆) in rice. The samples were ultrasonically extracted with 0.1% trifluoroacetic acid (TFA) containing 6.3 mmol/L diethylenetriaminepentaacetic acid (DTPA), and then the seven biothiols were derivatized with monobromobimane (mBrB) as derivatization agent in 4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid (HEPPS) buffer solution (pH 8.0). The separation was performed on an Agilent Eclipse Plus C₁₈ column (50 mm×4.6 mm, 5 μm) with gradient elution of 0.1%TFA solution (the pH value was adjusted to 2.5 with hydrochloric acid) and acetonitrile as mobile phases at a flow rate of 0.8 mL/min. The detection was performed at 380 nm for excitation and 470 nm for emission. The calibration curves of the seven biothiols showed good linearity in the concentration range of 0.7-100.0 mg/L with the correlation coefficients (r²)≥0.9991. The limits of detection were 0.03-0.20 mg/L. The recoveries of standard addition were in the range of 89.26%-99.42% with the relative standard deviations (RSDs, n=6) of 2.05%-5.87%. The method is sensitive, accurate, reproducible and suitable for the simultaneous determination of Cys, GSH, PC₂, PC₃, PC₄, PC₅ and PC₆ in rice.

Key words: biothiols, high performance liquid chromatography (HPLC), pre-column derivatization, rice